Primer Extension Assay for Low-Abundance Variant Detection
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Solution Overview
Problem
Current nucleic acid detection methods struggle to efficiently detect and quantify low-abundance nucleic acid variants, particularly in the presence of high-abundance variants, leading to challenges in accurately identifying genetic variations and mutations.
Innovation Solution
A method involving primer extension reactions with chain terminating reagents and 3′ to 5′ exonuclease activity is employed to differentiate and detect low-abundance nucleic acid variants by selectively extending and digesting oligonucleotides, allowing for the removal of unextended primers and enhancing the detection of low-abundance variants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional nucleic acid detection methods are used, then high-abundance variants can be detected, but low-abundance variants cannot be accurately detected due to masking by high-abundance variants
Solution Approach 1:
The method segments the detection process into distinct phases: selective extension of primers based on their ability to bind to low-abundance variants, followed by selective digestion of unextended primers. This segmentation allows the system to distinguish between primers that successfully extended (indicating low-abundance variant presence) and those that did not, thereby eliminating the masking effect of high-abundance variants.
Solution Approach 2:
The method extracts the critical function of primer extension to a specific enzymatic step, where only primers capable of binding to low-abundance variants are extended. Unextended primers are then removed through selective digestion. This extraction separates the detection signal (extended primers) from the background noise (unextended primers), enabling accurate detection of low-abundance variants.
2Measurement precision
If primer extension reactions are performed without selective digestion, then all primers remain in the reaction mixture, but this increases background noise and reduces detection accuracy
Solution Approach 1:
The method extracts unextended primers from the reaction mixture through selective digestion by exonuclease. This removal step eliminates the background noise generated by unextended primers, thereby improving detection accuracy by ensuring that only extended primers (which contain the detection signal) remain in the final analysis.
Solution Approach 2:
The method discards unextended primers through selective digestion by exonuclease, while recovering extended primers that contain the detection signal for low-abundance variants. This selective discarding and recovering process ensures that only relevant signals are analyzed, improving detection accuracy by eliminating irrelevant background noise.
3Adaptability or versatility
If multiple nucleic acid variants are detected simultaneously, then comprehensive analysis is achieved, but the complexity of the detection system increases
Solution Approach 1:
The method employs universal detection principles that can be applied to multiple nucleic acid variants simultaneously. The same primer extension and selective digestion mechanism works for detecting different variants, allowing multiplex detection without requiring separate specialized assays for each variant, thereby managing system complexity while achieving comprehensive analysis.
Solution Approach 2:
The method segments the detection process into modular steps that can be independently optimized and applied to multiple targets. Each variant detection follows the same segmented protocol (hybridization, selective extension, selective digestion), allowing comprehensive multiplex analysis while maintaining manageable system complexity through standardized procedures.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method enables sensitive detection and quantification of low-abundance nucleic acid variants, even when present at very low concentrations, by effectively distinguishing and analyzing extended oligonucleotides, thereby improving genetic variation analysis and mutation detection.
Implementation Method 1
hybridizing a target nucleic acid to an oligonucleotide that comprises a region that corresponds to a portion of the target nucleic acid, thereby generating a hybridized oligonucleotide
Implementation Method 2
contacting the hybridized oligonucleotide with an extension composition comprising one or more chain terminating reagents under extension conditions; thereby generating extended oligonucleotides
Implementation Method 3
contacting the products of (b) with an enzyme having 3′ to 5′ exonuclease activity, whereby unextended oligonucleotides are digested and extended oligonucleotides comprising a chain terminating reagent are not digested
Data Source
AI summary
Provided herein are products and processes for primer extension reactions using chain terminating reagents and enzymes having 3′ to 5′ exonuclease activity, whereby unextended oligonucleotides are digested and extended oligonucleotides are not digested. Also provided herein are products and processes for the detection of the presence, absence or amount of one or more variants of a target nucleic acid species or a plurality of target nucleic acid species comprising primer extension reactions using chain terminating reagents and enzymes having 3′ to 5′ exonuclease activity, whereby unextended oligonucleotides are digested and extended oligonucleotides are not digested.